Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins.

Ubiquitination is a key post-translational modification in the regulation of numerous biological processes in eukaryotes. The primary roles of ubiquitination are thought to be the triggering of protein degradation and the regulation of signal transduction. During protein ubiquitination, substrate specificity is mainly determined by E3 ubiquitin ligase (E3). Although more than 600 genes in the human genome encode E3, the E3s of many target proteins remain unidentified owing to E3 diversity and the instability of ubiquitinated proteins in cell. We demonstrate herein a novel biochemical analysis for the identification of E3s targeting specific proteins. Using wheat cell-free protein synthesis system, a protein array containing 227 human and 23 mouse recombinant E3s was synthesized. To establish the high-throughput binding assay using AlphaScreen technology, we selected MDM2 and p53 as the model combination of E3 and its target protein. The AlphaScreen assay specifically detected the binding of p53 and MDM2 in a crude translation mixture. Then, a comprehensive binding assay using the E3 protein array was performed. Eleven of the E3s showed high binding activity, including four previously reported E3s (e.g., MDM2, MDM4, and WWP1) targeting p53. This result demonstrated the reliability of the assay. Another interactors, RNF6 and DZIP3-which there have been no report to bind p53-were found to ubiquitinate p53 in vitro. Further analysis showed that RNF6 decreased the amount of p53 in H1299 cells in E3 activity-dependent manner. These results suggest the possibility that the RNF6 ubiquitinates and degrades p53 in cells. The novel in vitro screening system established herein is a powerful tool for finding novel E3s of a target protein

Rapid dissemination of alpha-synuclein seeds through neural circuits in an in-vivo prion-like seeding experiment

Abstract Accumulating evidence suggests that the lesions of Parkinson’s disease (PD) expand due to transneuronal spreading of fibrils composed of misfolded alpha-synuclein (a-syn), over the course of 5–10 years. However, the precise mechanisms and the processes underlying the spread of these fibril seeds have not been clarified in vivo. Here, we investigated the speed of a-syn transmission, which has not been a focus of previous a-syn transmission experiments, and whether a-syn pathologies spread in a neural circuit–dependent manner in the mouse brain. We injected a-syn preformed fibrils (PFFs), which are seeds for the propagation of a-syn deposits, either before or after callosotomy, to disconnect bilateral hemispheric connections. In mice that underwent callosotomy before the injection, the propagation of a-syn pathology to the contralateral hemisphere was clearly reduced. In contrast, mice that underwent callosotomy 24 h after a-syn PFFs injection showed a-syn pathology similar to that seen in mice without callosotomy. These results suggest that a-syn seeds are rapidly disseminated through neuronal circuits immediately after seed injection, in a prion-like seeding experiment in vivo, although it is believed that clinical a-syn pathologies take years to spread throughout the brain. In addition, we found that botulinum toxin B blocked the transsynaptic transmission of a-syn seeds by specifically inactivating the synaptic vesicle fusion machinery. This study offers a novel concept regarding a-syn propagation, based on the Braak hypothesis, and also cautions that experimental transmission systems may be examining a unique type of transmission, which differs from the clinical disease state

Summary of hit E3 ubiquitin ligases (E3s).

<p>E3s for which the relative luminescence signals in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156718#pone.0156718.g004" target="_blank">Fig 4A</a> were higher than 8 are listed. E3s indicated in bold character have been previously reported to interact with p53.</p

RNF6-dependent ubiquitination and degradation of p53 in cells.

<p>(A) Effect of RNF6 on p53 expression level. H1299 cells were co-transfected with a fixed amount of p53-V5 plasmid (25 ng) and an increased amount of FLAG-RNF6 plasmid (100 to 400 ng). Cell lysates from each reaction were subjected to SDS-PAGE followed by immunoblot analysis using anti-V5 antibody and anti-FLAG antibody. The band intensity of p53 in each reaction was quantified using imageJ software. Relative intensities normalized with the reaction of empty vector were indicated below the blot detected with anti-V5 antibody. (B) Co-transfection of p53-V5 with the wild-type (WT) or activity-deficient mutant (C/S) RNF6. (C) Stability of p53 in the presence of wild-type or C/S mutant of RNF6 was investigated by a pulse-chase assay. H1299 cells co-transfected with p53-V5 and wild-type or C/S mutant of FLAG-RNF6 were treated with 100 μg/ml of cycloheximide (CHX) for indicated time periods. The amount of p53-V5 detected by immunoblot analysis was quantified as same procedure as (A), and relative intensities normalized with the reaction without cycloheximide were indicated below the blot. (D) Endogenous RNF6 in H1299 cells were knockdown by siRNAs targeting RNF6, and overexpressed p53 was detected by immunoblot analysis (upper). The intensity of p53 band in each lane was quantified and intensites relative to the control were indicated below. Efficiency of RNF6 knockdown was confirmed with reverse transcription-quantitative PCR (lower).</p

Comprehensive screening to identify p53-binding E3 ligases.

<p>(A) AlphaScreen assay to detect the binding between p53 and 258 E3s including eight negative controls. In this experiment, binding of all E3s to biotinylated p53 and biotinylated DHFR was measured, and the relative value was calculated as follows: value from E3 and p53 / value from E3 and DHFR. Green arrowheads indicated the negative controls. (B) <i>In vitro</i> ubiquitination assay using p53-binding E3s obtained in (A). p53-V5 was mixed with each E3, and the ubiquitination assay was carried out using His-ubiquitin. Left panel, p53-V5 was precipitated with anti-p53 antibody and detected with anti-ubiquitin antibody. Right panel, His-ubiquitin was pull-down with Ni-sepharose, and ubiquitinated p53 was detected with anti-p53 antibody.</p

Model assay using p53 and MDM2.

<p>(A) Expression of recombinant proteins of p53 and MDM2 with the wheat cell-free system. Wild-type (WT) and mutant p53 lacking the amino acid residues required for binding with MDM2 (∆I) were synthesized as N-terminal biotinylated form. Wild-type MDM2 and its mutant with a single amino acid substitution in the catalytic core cysteine residue (C/S) were synthesized as N-terminal FLAG-tagged protein. Biotinylated and FLAG-dihydrofolate reductase (FLAG-DHFR) were used as negative controls for p53 and MDM2, respectively. The total translation mixture of each protein was centrifuged, and the whole translation mixture (W) and supernatant (S) were subjected to SDS-PAGE followed by immunoblot analysis (IB) using the antibodies indicated below. (B) Binding assay of biotinylated p53 and FLAG-MDM2 using a conventional immunoprecipitation (IP) assay. Fifteen microliters of crude biotinylated p53 (WT or ∆I) and FLAG-MDM2 were mixed and precipitated with anti-FLAG-antibody. Immunopreciptates were detected with anti-FLAG antibody and anti-biotin antibody. FLAG-DHFR was used as controls for FLAG-MDM2. (C) <i>In vitro</i> ubiquitination assay using biotinylated p53 and FLAG-MDM2. The crude translation mixtures of biotinylated p53 (WT or ∆I) and FLAG-MDM2 (WT or C/S) were mixed and then the reaction mixture containing ATP and HA-tagged ubiquitin (HA-Ubi) with or without UbcH5b was added. After the ubiquitination reaction, biotinylated p53 was pulled down with streptavidin (STA) magnet beads and subjected to SDS-PAGE followed by immunoblot analysis using anti-HA-antibody (for ubiquitin) and anti-biotin antibody (for p53).</p